CDNA Cloning,Expression And Characterization Of Glucose Oxidase From Heliothis Viriplaca

Glucose oxidase(GOX, EC1.1.3.4) is an important oxidoreductase in living organism. It has specificity of hydrolysis glucose. It can be targeted to β-D-glucose, and decompose glucose to gluconic acid and hydrogen peroxide. These two substances continue to participate in other important metabolic pathways. Therefore, glucose oxidase in plants, animals and microorganisms are the most important functional enzyme.Heliothis viriplaca belongs to Noctuidae, Lepidoptera. It is a kind of common soybean pest. It often feeds on the soybean leaves and young tissues, and damages the soybean pods. Finally, it effects soybean yield reduction and quality deterioration. H. viriplaca not only damages field soybean, but also harms alfalfa, cotton, flax and other crops.Total RNA was isolated from the H. viriplaca. The glucose oxidase c DNA sequence was cloned by RT-PCR and Rapid amplification of c DNA ends technique. The c DNA sequence was expressed by the recombinant prokaryotic expression vector p ET21 b in E. coli. The recombinant protein was purified by Ni-NAT affinity chromatography. After being purified, the recombinant protein was renatured using gradient dialysis technique. The activity of GOX protein was determined with glucose as the substrate. The bacteriostasis function of the glucose oxidase in the partial bacteria was explored by inhibitory zone with filter paper. The transcriptional level of GOX gene in different tissues of H. viriplaca, transcriptional response of GOX gene to different concentrations of glucose and transcriptional response of GOX gene to starvation and feeding w ere carried out by Quantitative real-time PCR technique. The main research findings are as follows:(1) The c DNA sequence of Hv GOX was 2154 bp, including an open reading frame of 1824 bp encoding a polyeptide of 607 amino acids with an estimated molecular ma ss of 67.04 KDa and an estimated isoeletric point of 5.13. The c DNA sequence has been deposited in Gene Bank with accession No. KT907054 and designated as Hv GOX. Multiple sequence alignment indicated that Hv GOX shared 60, 89 and 76% amino acid sequence identity with GOX from Helicoverpa armigera(Gene Bank accession No.ACC94296). Helicoverpa zea(Gene Bank accession No.ACJ71598) and Spodoptera exigua(Gene Bank accession No. ADL38963). All the results indicated that a novel glucose oxidase gene was cloned from H. viriplaca.(2) Hv GOX was cloned into prokaryotic expression vector p ET21 b and expressed in BL21 competent cells. By the induction of IPTG, the recombinant protein was successfully expressed in E. coli. SDS-PAGE results revealed that recombinant protein molecular weight consisted with the prediction of protein molecular weight. Because the expression recombinant protein was inclusion body protein, it was actived after denaturation, purification and renaturation. The activity of renatured recombinant protein and commercialized protein were measured with glucose as the substrate. The measurement results showed that the activity of recombinant protein was lower than commercialized protein. It was only 1/16 times. Zone of inhibition to bacteria test performed with the same concentrations of both proteins. The results revealed that prokaryotic expressed GOX had low activity, but commercialized protein had a good antibacterial effect.(3) Real-time PCR results showed that the m RNA transcripts were expressed in d ifferent tissues, and the highest expression level was in salivary gland. Using soybean leaves soaked by 0.01%, 0.1%, 1% and 10% glucose solution to feed larvae, the GOX expression levels in the larvae were all induced, and the expression level of the concentration of 10% was the highest. When larvae of H. viriplaca at 4th instar were starved for 3, 6, 12, 24, 48 h, the results showed that the expression level in the starvation of 12 h was the highest.(4) The technique of RNAi research of Hv GOX gene showed that, after the H.viriplaca larvae at 5th instar injected with ds RNA 2 μL at the concentration of 2 μg/μL, the transcriptional levels of Hv GOX gene significantly decreased after 24 h treatment. The results showed that efficiency of RNAi-mediated knockdown of Hv GOX gene was successful.